Proteases for

biocatalysis
for smarter chemical synthesis
Biocatalysis
Biocatalysis involves the implementation of natural catalysts, such as enzymes, in place of chemical catalysts in synthetic
processes. Compared to chemical catalysts, enzymes offer:

• higher reaction rates

• milder reaction conditions
• high reaction specificity with no side products

This change can enable new, more sustainable routes for the production of intermediates and active pharmaceutical ingredients
(APIs). However, please note Novozymes products do not comply with manufacturing according to pharmaceutical standards and
Novozymes products must not be used as active pharmaceutical ingredients (APIs) or excipients.

Biocatalysis has become an increasingly important tool for medicinal, process and polymer chemists, allowing the development
of efficient and highly attractive synthetic processes on an industrial scale. Use of enzymes in catalysis is a well-established
technology within the chemical industry. An advantage of enzymes in organic synthesis is their remarkable selective properties,
which provide commercial benefits including:

• high selectivity in production of single stereoisomers

• fewer side reactions
• less reprocessing and purification steps
• easier product separation
• less pollution

The combination of all of these advantages leads to a reduction in costs.

Enzyme catalysts work by lowering the activation energy (Ea‡) for a reaction, thus dramatically increasing the rate of the
reaction. As a result, products are formed faster and reactions reach their equilibrium state more rapidly. Most enzyme reaction
rates are millions of times faster than those of comparable uncatalyzed reactions. As with all catalysts, enzymes are not
consumed by the reactions they catalyze, nor do they alter the equilibrium of these reactions. However, enzymes do differ from
most other catalysts in that they are highly specific for their substrates.

C E
A

A. Various microorganisms can D

be used to produce natural catalysts
such as enzymes
B. The enzymes are separated from the
microorganisms and subsequently
partially purified and formulated
C. An enzyme attracts specific
substrates to its active site B

D. It catalyzes the chemical reaction by

which products are formed
E. It then allows the products
to separate from the enzyme surface
Proteases
Proteases (EC 3.4.21.62) are enzymes which conduct proteolysis by hydrolysis of the peptide bonds that link amino acids
together in the polypeptide chain forming the protein. Proteases belong to the class of enzymes known as hydrolases catalyzing
the reaction of hydrolysis of various bonds with the participation of a water molecule.

Proteases can be used in organic synthesis to resolve a pair of enantiomeric forms in racemic mixtures through kinetic
resolution where one enantiomer in the mixture is more rapidly transformed than the other.

Protease catalysts can resolve enantiomers through a variety of reactions such as:

• hydrolysis of esters or amides of carboxylic acid

• esterification or transesterification reactions
• amide/peptide bond formation

Proteolysis of a peptide bond:

Key applications of proteases

Kinetic resolution of amino acids by hydrolysis of racemic amino esters which can be converted into dynamic kinetic resolution
by addition of catalytic aldehyde1:

Kinetic resolution of carboxylic acid by hydrolysis of carboxylic ester:

Kinetic resolution of amino acid by hydrolysis of racemic amino ester:

Other potential applications for proteases include:

• hydrolysis of selective amides2

• formation of low molecular weight peptides3
• transesterifications

Serine proteases
Serine proteases contain a serine group in their active site which is essential for substrate binding and cleavage. Serine
proteases are characterized by their broad substrate specificity and their activity extends beyond purely peptidase to include
esterase and amidase activities. The common reaction mechanism is in the form of a catalytic center containing serine as a
nucleophile, aspartate as an electrophile and histidine as a base. The reaction mechanism involves the formation of covalently
linked enzyme substrate intermediate through acylation resulting in loss of the corresponding amino acid or peptide fragment.
Nucleophilic attack on the intermediate by water results in deacylation thereby completing hydrolysis of the peptide.
Subtilisin A
Subtilisin A (E.C. 3.4.21.62) is an alkaline non-specific serine protease from Bacillus subtilis that initiates the nucleophilic attack
on the peptide bond through a serine residue at the active site; it catalyzes the hydrolysis of proteins and peptide amides.

Alcalase®
Alcalase® acts as an esterase, enabling it to catalyze stereoselective hydrolysis of some esters. Alcalase also efficiently
hydrolyzes amino esters which include heterocyclic amino esters.

Savinase®
Savinase® catalyzes stereoselective hydrolysis of some esters as well as strained amides under alkaline conditions.

Esperase®
Esperase® is an endo-peptidase with a broad specificity which performs well in alkaline conditions and at elevated
temperatures as compared to other microbial serine proteases.

Kinetic resolution of carboxylic acids by hydrolysis of carboxylic esters4:

Kinetic resolution of strained amides2:

Stability of proteases
The graphs below represent alcalase pH and temperature stability.

Effect of pH on alcalase activity Effect of temperature on alcalase activity Effect of pH on alcalase stability

100 100 100

80 80 80

60 60 60
Relative activity (%)

Relative activity (%)

Relative activity (%)

40 40 40

20 20 20

0 0 0
4 5 6 7 8 9 10 10 11 12 10 20 30 40 50 60 70 80 90 100 3 4 5 6 7 8 9 10 11 12

pH Temperature (°C) pH
Neutrase®
Neutrase® (E.C.3.4.24) is a neutral, zinc metallo endo-protease from Bacillus amyloliquefaciens that randomly hydrolyses
internal peptide bonds and also facilitates enzymatic synthesis of oligopeptides by the reverse proteolysis reaction with zinc
metal as co-catalyst. Neutrase® belongs to the same protease family as thermolysin, a zinc dependent metallo
endo-protease.

Thermolysin in an immobilized form has been used successfully in industrial processes for synthesis of an Aspartame
intermediate5. The reaction takes place in organic solvent and involves kinetic resolution of an amine methyl ester with high
enatioselectivity and high regioselectivity in the amide bond formation of the a-carbonyl in Aspartic acid preferred over the
b-carbonyl.

rTrypsin®
rTrypsin® (EC 3.4.21.4) is an endopeptidase that preferentially hydrolyses ester bonds whose carboxyl groups are contributed
by lysine (Lys) or arginine (Arg) except when either is followed by proline. The enzymatic mechanism of action is similar to other
serine proteases. The aspartate residue located in the catalytic pocket of rTrypsin is responsible for attracting and stabilizing
positively charged lysine and/or arginine, and is thus responsible for the specificity of the enzyme.

Benefits of enzymes as biocatalysts in organic transformations

Cost savings Improved productivity Improved quality of API/intermediate Environmental friendliness

• reduction in raw material input
• avoids use of costly chiral
resolving agents or costly metal
based catalysts
• lower equipment, labor and
energy costs
• shortened synthesis routes
• more batches resulting in increased
capacity
• avoids laborious protection and
de-protection
• higher yields
• fewer or no by-products, leading • reduction of waste products
to reduced impurities in the final produced and solvent usage
products • higher energy savings
• high stereo-, regio-, and chemo-
selectivity
• less residual solvent carry over from
reduced solvent use

Available from Novozymes

Product EC Optimum usage
Type Form Activity Applications
name number conditions

Alcalase 3.4.21.62 Serine endo-peptidase Liquid 30–65°C, 2.4 AU-A/g Stereoselective hydrolysis of amino esters
2.4 L FG (mainly subtilisin A) pH 7–9 and selective esters

Alcalase 3.4.21.62 Serine endo-peptidase Liquid 30–65°C, 2.5 AU-A/g Stereoselective hydrolysis of amino esters
2.4 L, DX (mainly subtilisin A) pH 7–9 and selective esters

Savinase 3.4.21.62 Serine endo-peptidase Granulate 30–70°C, 12 KN-PU- Stereoselective hydrolysis of amino esters
12 T (mainly subtilisin A) pH 8–10 S/g and selective esters

Savinase 3.4.21.62 Serine endo-peptidase Liquid 30–70°C, 16 KN-PU- Stereoselective hydrolysis of amino esters
16 L (mainly subtilisin A) pH 8–10 S/g and selective esters

Esperase 3.4.21.62 Serine endo-peptidase Liquid 30–70°C, 8 KNPU-E/g Serine endoprotease that hydrolyzes internal
8.0 L (mainly subtilisin A) pH 8–10 peptide bonds

Neutrase 3.4.24.28 Metalloprotease Liquid 40–50°C, pH 7 0.8 AU-N/g Metallo endoprotease that hydrolyz-es internal
0.8 L peptide bonds

rTrypsin 3.4.21.4 Serin protease Granulate pH 7.8–8.0 800 USP/mg Hydrolysis of amid end ester bonds of lysine
and arginine at carboxyl terminal

* K = Kilo, AU = Anson Unit, NPU = Novo Protease Unit, 1 AU = 1NPU, ASNU = Asparaginace Unit, USP = Trypsin activity unit using USP Crystallised Trypsin
Reference Standard. The activity is determined relative to a protease A standard. The result is given in the same units as the standard.
1 ASNU is the amount of enzyme that produces 1 µmol Ammonia per minute under the standard reaction conditions.
References
1. Pietruszka, J., Simon, R.C., Kruska, F., and Braun, M. (2010) Euro.J.Org.Chem., 6217-6224
2. Schonherr, H., Mollitor, J., and Schneider, C.(2010) Eur.J.Org.Chem., 20, 3908-3918
3. Gedey, S., Lijebald, A., Lazar, L., Fulop, F., and Kanerva, L.T.,(2002), Can.J, Chem., 80, 565-570
4. Ref. Carlos A. Martinez, Shanghui Hu, Yves Dumond, Junhua Tao, Patrick Kelleher, Liam Tully Organic Process
Research & Development 2008, 12, 392-398
5. Hari Krishna, S., Persson, M., and Bornscheuer, U.T. (2002) Tetrahedron Asymmetry, 13, 2693-269
6. Ozdermirhan, D., Sezer, S., and Sonmez, Y.(2008) Tetrahedron Asymmetry ,19, 2717-2720.

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June 2016 - No. 2014-12715-04

Novozymes does not promote or support the use of enzymes as Active Pharmaceutical Ingredients (APIs) or excipients.
Novozymes enzymes are industrial bulk products and are not produced according to pharmaceutical standards. If enzymes
are used as process aids, it is the responsibility of the API or excipient manufacturer to assure that the enzymes are suitable
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for the intended use, including but not limited to evaluation of purity and absence of carry over in the final product. Laws,
regulations, and/or third party rights may prevent customers from importing, using, processing, and/or reselling the products
described herein in a given manner.
Without separate, written agreement between the customer and Novozymes to such effect, this document does not
constitute a representation or warranty of any kind and is subject to change without further notice.